Rabbit anti mouse phospho nf κb p65 antibody

First, the lung tissues of the mice in each group were rapidly ground in liquid nitrogen, and then the samples were transferred to prechilled 1.5 mL centrifuge tubes and lysed using a RIPA lysis solution (Solarbio, China) containing protease inhibitors and phenylmethylsulfonyl fluoride (PMSF) for 30 min, and the cellular proteins were extracted. After the protein concentration was determined by the BCA method, equal amounts of proteins from each group were loaded for SDS-PAGE and then transferred to a PVDF membrane. The membrane was blocked with 5% fat-free milk for 2 h. The membrane was incubated with a rabbit anti-mouse NF-κB p65 antibody (1:1,000; Cell Signaling Technology, USA), rabbit anti-mouse phospho-NF-κB p65 antibody (1:1,000; Cell Signaling Technology), and rabbit anti-mouse β-actin antibody (1:5,000; Proteintech, USA) for 2 h at room temperature. Then, the membrane was incubated with goat anti-rabbit IgG DyLight 800 antibody (1:30,000; Cell Signaling Technology) for 1 h at room temperature in the dark. Protein expression was detected using a fluorescence imager (LI-COR, USA), and relative gray values were calculated.